Study On The Preparation And Application Of Novel DNA Biosensors

With the deeper research on the structure and function of human gene, especially the fast progress of the Human Genome Project, the study on DNA diagnosis and gene treatment of human disease has been greatly promoted. Many genes inducing disease were discovered and identified. Little changes in DNA sequence, such as base-pair displacement, lack and manifold can lead to the disorder of human inherited character and various diseases. Therefore, much effort is needed to detect the specific DNA sequences related to diseases, which is also helpful for the selection of genes and the development of drugs, clinical diagnosis, epidemic prevention, environmental protection and bioengineering.Traditional DNA probes are labeled with radioelements. Although this method gives high sensitivity, there are many problems such as a potential hazard to analyst environment, and time-consuming because of radioactive labels. In order to solve these problems, a series of non-radioactive DNA probes such as fluorescent, chemiluminescent and electrochemical probes have been developed. Among these, electrochemical DNA biosensor is a novel technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and it can be compatible with DNA biochip. DNA biosensor based on Fluorescent detection method is widely used in gene sequence detection, antibody immune analysis, diseases diagnosis and antitumor drug study for its speediness, repeatability and non-radioactivity.This dissertation focuses on studing novel DNA biosensors with remarkable sensitivity and selectivity. We developed electrochemical biosensor based on magnetite nanoparticles by electrochemical technique combined with DNA hybridization technique and nanotechnique; Adoping homogeneous assay andfluorescence quenching method, a very simple and convenient protocol for DNA hybridization and mismatched mutation sequence detection is proposed.The dissertation includes four chapters:Chapter 1 PrefaceFirstly, the progress, the principle( DNA sequence identification principle and immobilization method of ssDNA on solid support) and the classification of DNA biosensor were introduced; The characters, application and development trends of DNA biosensor were summarized. Secondly, magnetic nanoparticles and its application in gene separation and detection were introduced. Thirdly, fluorescent probe and its application were described. At last, we pointed out the purpose and significance of the dissertation.Chapter 2 A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti walled carbon nanotubes in polypyrroleIn this new detection system, mercapatoacetic acid (RSH)-coated magnetite nanoparticles, capped with 5'-(NH2) oligonucleotide, is used as DNA probe to complex 29-base polynucleotide target (a piece of human porphobilinogen deaminase PBGD promoter from 170 to 142). Target sequence hybridized with the probe results in the decrease of the reduction peak current of daunomycin connected with probe. The response of non-complementary sequence was almost the same as the blank, and the response of three-base mismatched sequence within 29-base polynucleotide was obviously distinguished from complementary sequence, which can easily identify point mutation of DNA. The equation of calibration plot is ip (uA) = 0.8255 -0.0847cdnax10'3 in the range of 6.9* 10'14 ~ 8.6* 10"13 mol/L, and correlation coefficient is 0.9974. The detective limit is 2.3 xio14 mol/L of target oligonucleotide. This device can be optimized for the detection of complex sequence.Chapter 3 Magnetic microsphere coated with polymer as oligonucleotides support for electrochemical stripping detection of DNA hybridizationMagnetic microsphere coated with polymer is a kind of affinity solid support, which has small particle size, big specific surface area and paramagnetism. In this paper, we adopted coprecipitation method to prepare Fe3O4 magnetite nanoparticle by m...